Production of ectomycorrhizal mycelium peaks during canopy closure in Norway spruce forests

Abstract

Here, species composition and biomass production of actively growing ecto-mycorrhizal (EM) mycelia were studied over the rotation period of managed Norway spruce (Picea abies) stands in southwestern Sweden. • The EM mycelia were collected using ingrowth mesh bags incubated in the forest soil during one growing season. Fungal biomass was estimated by ergosterol analysis and the EM species were identified by 454 sequencing of internal transcribed spacer (ITS) amplicons. Nutrient availability and the fungal biomass in soil samples were also estimated. • Biomass production peaked in young stands (10–30 yr old) before the first thinning phase. Tylospora fibrillosa dominated the EM community, especially in these young stands, where it constituted 80% of the EM amplicons derived from the mesh bags. Species richness increased in older stands. • The establishment of EM mycelial networks in young Norway spruce stands requires large amounts of carbon, while much less is needed to sustain the EM community in older stands. The variation in EM biomass production over the rotation period has implications for carbon sequestration rates in forest soils.

Full text

Production of ectomycorrhizal mycelium peaks during
canopy closure in Norway spruce forests
Ha
˚kan Wallander
1
, Ulf Johansson
2
, Erica Sterkenburg
3
, Mikael Brandstro
¨m Durling
3
and Bjo
¨rn D. Lindahl
3
1
Department of Microbial Ecology, Lund University, SE–223 62 Lund, Sweden;
2
Swedish University of Agricultural Sciences, To
¨nnersjo
¨heden
Experimental Forest, PO Box 17, SE–31038 Simla
˚ngsdalen, Sweden;
3
Department of Forest Mycology and Pathology, Swedish University of Agricultural
Sciences PO Box 7026, SE–750 07 Uppsala, Sweden
Author for correspondence:
Ha
˚kan Wallander
Tel: +46 46 2223759
Email: hakan.wallander@mbioekol.lu.se
Received: 10 March 2010
Accepted: 4 May 2010
New Phytologist (2010) 187: 1124–1134
doi: 10.1111/j.1469-8137.2010.03324.x
Key words: 454 sequencing,
chronosequence, ectomycorrhiza, ergosterol,
external mycelia.
Summary
•Here, species composition and biomass production of actively growing ecto-
mycorrhizal (EM) mycelia were studied over the rotation period of managed
Norway spruce (Picea abies) stands in south-western Sweden.
•The EM mycelia were collected using ingrowth mesh bags incubated in the
forest soil during one growing season. Fungal biomass was estimated by ergosterol
analysis and the EM species were identified by 454 sequencing of internal
transcribed spacer (ITS) amplicons. Nutrient availability and the fungal biomass in
soil samples were also estimated.
•Biomass production peaked in young stands (10–30 yr old) before the first
thinning phase. Tylospora fibrillosa dominated the EM community, especially in
these young stands, where it constituted 80% of the EM amplicons derived from
the mesh bags. Species richness increased in older stands.
•The establishment of EM mycelial networks in young Norway spruce stands
requires large amounts of carbon, while much less is needed to sustain the EM
community in older stands. The variation in EM biomass production over the
rotation period has implications for carbon sequestration rates in forest soils.
Introduction
Nutrient uptake of boreal forest trees is mainly mediated
through symbiotic ectomycorrhizal (EM) fungi, which
colonize the tree roots producing an extensive network of
external mycelium in the soil (Smith & Read, 2008). In
addition to its importance for nutrient uptake, EM myce-
lium serves as an important food source for soil animals and
saprotrophic microorganisms, and in this way fuels the soil
ecosystem with energy derived from the photosynthetic
activity of the trees. These EM mycelia can also contribute
significantly to soil respiration; up to 50% of soil respiration
has been attributed to roots and their associated EM fungi
(Ho
¨berg et al., 2001). The large proportion of photo-
synthetic activity that is allocated to EM fungi (10–50%,
reviewed by Simard et al., 2002) indicates a large potential
for EM fungi to influence carbon (C) flux below ground
and consequently the sequestration of carbon in the soil.
The allocation of C to fine roots in managed forest soils
has been shown to vary over the rotation period, with a
distinct maximum in young stands (King et al., 2007). The
amount of C allocated to EM fungi over the same period
may follow a similar pattern, as the growth of fine roots is
positively correlated with that of EM mycelium (Majdi
et al., 2008). Maximum belowground C allocation appears
to coincide with intense nutrient demand at canopy closure,
when the leaf area index is at a maximum (Simard et al.,
2002), which is reached between 25 yr and 40 yr in
Norway spruce (Picea abies) forests in south-central Sweden
(Schmalholz & Hylander, 2009). However, studies on the
production of EM fruit bodies in forests of different ages
have provided inconclusive results. Some studies report a
peak in production in young forest (Chisilov & Demidova,
1998; Hintikka, 1988), while others report no variation
with forest age (Bonet et al., 2004).
Ectomycorrhizal fungal communities in boreal forests are
highly diverse (Dahlberg, 2001), and successions of EM
communities over the rotation period of different types of
forest ecosystems are well documented from studies of
sporocarps or root tips (Kranabetter et al., 2005; Palfner
et al., 2005; Twieg et al., 2007). Successional change in
species composition have been explained by changes in soil
New
Phytologist
Research
1124 New Phytologist (2010) 187: 1124–1134
www.newphytologist.com
The Authors (2010)
Journal compilation New Phytologist Trust (2010)

chemistry such as pH and nitrogen (N) availability, and the
chemical characteristics of the organic material, which usu-
ally becomes more recalcitrant as the forest ages (Deacon &
Flemming, 1992; Jumpponen et al., 1999). In addition, a
variation in C delivery by the host may influence EM suc-
cession, as indicated by the observed changes in EM com-
munity composition in response to elevated CO
2
concentrations (Fransson et al., 2001; Parrent & Vilgalys,
2007). In earlier research, EM fungi have been described in
terms of early- and late-stage fungi based on their occur-
rence with forest age (Last et al., 1987; Deacon &
Flemming, 1992), but these concepts have been criticized
in more recent studies for being insufficient to describe
fungal successions in forest ecosystems (Kranabetter et al.,
2005; Palfner et al., 2005; Twieg et al., 2007).
In the present study, the aim was to estimate production
of EM biomass, as extraradical mycelium, over a Norway
spruce chronosequence ranging from 0 to 130 yr of age in
south-western Sweden. In addition, we analysed how the
EM fungal community changed over this period, using
novel high-throughput 454 sequencing (Margulies et al.,
2005; Bue
´eet al., 2009) of internal transcribed spacer (ITS)
amplicons. We used sand-filled ingrowth mesh bags to esti-
mate EM production and to identify the active, extraradical
EM community in the soil (Wallander et al., 2001; Hedh
et al., 2008). This approach has been used in a number of
studies for both quantifying EM growth in soil and for anal-
ysing changes in EM communities in response to nutrient
amendments or forest management practices (Kjo
¨ller,
2006; Korkama et al., 2007; Parrent & Vilgalys, 2007;
Hedh et al., 2008; Majdi et al., 2008). A peak in EM
growth was expected during canopy closure. We also
expected changes in EM community composition with
stand age, owing to shifting host C allocation patterns, soil
chemical parameters and organic matter quality.
Materials and Methods
Study sites
The sites studied are located in the To
¨nnersjo
¨heden
Experimental Forest (lat. 5642¢N, long. 1306¢E, alt. 60–
140 m above sea level) in south-western Sweden
(Malmstro
¨m, 1937), representing maritime climate condi-
tions (yearly mean air temperature +6.7C, yearly mean
precipitation 1064 mm, mean length of growing season
215 d). Forty Norway spruce (Picea abies (L.) H. Karst)
stands were selected with the ages evenly distributed
between 0 and 130 yr (see the Supporting Information
Table S1). The different stands represented the following
forest development classes: fresh and recently cultivated
clearcuts (n= 10), young stands before first thinning
(n= 10), stands in the active thinning phase (n= 10) and
old stands after the active thinning phase (n= 10). Each
class was further divided into one younger and one older
subclass, resulting in eight different classes with five stands
in each. All sites were located on mineral soils representing
the soil types till or gravel. The type of former land use at
the sites was varied, representing first and second rotation of
pure Norway spruce stands following coniferous-dominated
stands, broadleaf-dominated stands and former open Calluna
heathland (Table S1).
Experimental design
To estimate the growth of EM fungi we used fungal
ingrowth bags made of nylon mesh (50 lm mesh size,
8·8·1 cm). This mesh size allows the ingrowth of fun-
gal hyphae, but not roots (Wallander et al., 2001). The
mesh bags were filled with 30 g acid-washed quartz sand
(0.36–2.0 mm, 99.6% SiO
2
; Ahlsell AB, Malmo
¨, Sweden).
Five replicate mesh bags were buried at the interface
between the organic horizon and the mineral soil in each
spruce stand (a total of 5 ·40 = 200 bags). The mesh bags
were placed in a row, c. 1 m apart, on 1 June 2005. The
mesh bags were harvested on 15 November 2005, after an
incubation period of 22 wk. Soil samples were collected
from the humus layer (c. 3–10 cm) and from the upper
region (0–5 cm) of the mineral soil using a soil corer (4 cm
diameter). Five cores, taken at random locations (c.1–2m
away from the location of the mesh bags) within each site
were pooled to give one composite sample.
The mesh bags were stored at +8C for up to 6 h before
being taken to the laboratory. The sand from all five mesh
bags from each site was combined and mixed, and a sub-
sample of 10 g was taken for the analysis of ergosterol as an
indicator of fungal biomass (see later). Another subsample
of 10 g was gently shaken with water in a glass flask to
loosen the sand from the fungal mycelium. The procedure
was repeated until the water was clear and no apparent
mycelium was visible in the water. The mycelium was
collected on a nylon mesh, and it was assumed that insignif-
icant amounts of hyphae were retained on the sand parti-
cles. The mycelium was collected in Eppendorf tubes and
the approximate volume was recorded by comparing with
an Eppendorf tube with following markings: 20, 60, 150,
300 and 500 ll. This sample was then used for DNA
extraction and identification of the EM community. The
samples were stored at )20C and freeze-dried until analy-
sed.
DNA extraction, PCR and 454 sequencing
The mycelium samples were mixed with Al
2
O
3
powder to a
total volume of c. 200 ll and ground together with glass
beads in a Fast Prep shaker (FP120; MP Biomedicals,
Irvine, CA, USA). Between 50 mg and 100 mg of this
mixture was then used for DNA extraction with 1 ml
New
Phytologist Research 1125
The Authors (2010)
Journal compilation New Phytologist Trust (2010)
New Phytologist (2010) 187: 1124–1134
www.newphytologist.com

extraction buffer (3% cetyl trimethylammonium bromide
(CTAB), 2 mM EDTA, 150 mM Tris-HCl and 2.6 M
NaCl, pH 8) at 65C for 1 h, followed by precipitation
with 1.5 volumes of isopropanol. After centrifugation
(15 min, 16 200 g ) the pellet was resuspended in 50 ll
Milli-Q water.
Polymerase chain reaction was carried out in two steps.
First, the fungi-specific primer combination ITS1-F
(Gardes & Bruns, 1993) and ITS4 (White et al., 1990)
were used at an annealing temperature of 57C for 35
cycles. In the second step, the same ITS primers were used,
but elongated with the adaptors required for 454 sequenc-
ing (ITS1-F ⁄A adaptor and ITS4 ⁄B adaptor) (Margulies
et al., 2005). The primers also contained sample-specific
tags consisting of five bases (Acosta-Martinez et al., 2008).
For the ITS1-F ⁄A adaptor primer, a single tag was used for
all samples (5¢-GCCTCCCTCGCGCCATCAGACCTG
CTTGGTCATTTAGAGGAAGTAA-3¢), while for the
ITS4 ⁄B adaptor primer, unique base combinations were
used for each individual sample (5¢-GCCTTGCCAGCC
CGCTCAGXXXXXTCCTCCGCTTATTGATATGC-3¢).
The same annealing temperature as above was used in this
second step, but for only five cycles. Between and after the
two PCR steps, products were purified with the Agencourt
AMPure kit (Agencourt Bioscience Corporation, Beverly,
MA, USA), in order to remove residual primers and primer
dimers.
The concentrations of the purified PCR products were
measured with the PicoGreen ds DNA Quantification Kit
(Molecular Probes, Eugene, OR, USA) on a luminescence
spectrometer (model LS50B; Perkin Elmer, Waltham, MA,
USA). Equal amounts of DNA from each sample were
pooled to provide a single sample for the entire study. The
454 sequencing was performed on a Genome Sequencer
FLX 454 (Roche Applied Biosystems) at the Department
of Biotechnology, the Royal Institute of Technology,
Stockholm, Sweden. A half-plate was sequenced, starting
from the ITS4 ⁄B adapter, providing partial coverage (200–
300 base pairs) of the fungal ITS regions.
Bioinformatic analysis of sequence data was conducted
using the SCATA software (http://scata.mykopat.slu.
se). Sequences were filtered for quality, removing short
sequences (< 200 bases), sequences with low quality (aver-
age read quality < 20 or > 5 bases with base quality < 10)
as well as primer dimers and polymers. Primer and sample
tag sequences were then removed, but the sample associa-
tion was stored as meta-data associated with each sequence.
All sequences were then searched against each other, using
blastn from the NCBI blast package (Altschul et al.,
1997) requiring a minimum match length of 190 bp.
Sequences were brought together in clusters when the
sequence similarity, excluding gaps, exceeded 98.5% (single
linkage clustering), resulting in a matrix of cluster relative
abundances vs sample identities. Within each cluster,
sequences were realigned, using the global alignment pro-
gram muscle (Edgar, 2004), and consensus sequences were
inferred. Clusters were taxonomically identified manually
by aligning consensus sequences together with selected
reference sequences from NCBI Genbank or the UNITE
database (Ko
¨ljalg et al., 2005), using the CLUSTALW
algorithm of megalign (DNAStar Inc., Madison, WI, USA).
Ergosterol analysis
Freeze-dried sand from the mesh bags was analysed for
ergosterol to provide an estimate of the EM fungal biomass
(Nylund & Wallander, 1992). Ergosterol was also used as
an estimate of fungal biomass in soil samples. Total ergo-
sterol (including esterified forms) is most commonly used
for fungal biomass estimates in soil, but recently it has been
suggested that free ergosterol is a better proxy for living
fungi (Yuan et al., 2008). We used one subsample (10 g
sand or 1 g soil) for estimates of free ergosterol, and one
subsample for total ergosterol. The free ergosterol was
extracted in 5 ml methanol, while the total ergosterol was
extracted with 5 ml 10% KOH in methanol. After this
step, the two methods followed the same protocol. The
samples were sonicated for 15 min, extracted overnight and
then refluxed at 70C for 90 min. After cooling, 1 ml
H
2
O and 2 ml cyclohexane were added. The samples were
mixed in a vortex apparatus for 20 s, centrifuged for 5 min
at 900 g and the cyclohexane phase was then transferred to
another test tube. The methanol was extracted with a fur-
ther 1.5 ml cyclohexane. The cyclohexane was evaporated
under N
2
and the samples were dissolved in methanol.
Before the quantification of ergosterol, the samples were fil-
tered through a 0.5 lm Teflon syringe filter (Millex LCR-
4; Millipore). The chromatographic system consisted of a
high-performance liquid chromatograph (Hitachi model
L2130, Japan), a UV detector (Hitachi model L2400,
Japan) and a C
18
reversed-phase column (Chromolith,
Merck) preceded by a C
18
reversed-phase guard column
(Elite LaChrome; Hitachi). Extracts were eluted with meth-
anol at a flow rate of 1 ml min
)1
and absorbance measured
at 282 nm.
Soil chemical analysis
The most available parts of NH
4
, calcium (Ca), potassium
(K) and magnesium (Mg) in the soil samples from the
humus layer were estimated by BaCl
2
extraction.
Subsamples of 25 g of soil were extracted with 100 ml
0.1 M BaCl
2
for 1 h. The pH of the solutions was
measured with a pH meter and the concentrations of the
elements were analysed using Inductively Coupled Plasma -
Emission Spectroscopy.
The most available proportion of phosphorus (P) of the
same samples was estimated by sulphate fluoride extraction.
1126 Research
New
Phytologist
The Authors (2010)
Journal compilation New Phytologist Trust (2010)
New Phytologist (2010) 187: 1124–1134
www.newphytologist.com

Subsamples of 15 g were extracted for 1 h in 70 ml
0.05 M Na
2
SO
4
and 0.02 M NaF. The solutions were
filtered through a 25 mm RC filter (Lida Manufacturing
Corp., Windsor, UK) and were stored at )20C until
analysed. The phosphate concentration in the filtrate was
determined using SnCl
2
as a reducing agent (Murphy &
Riley, 1962).
The C : N ratio was measured with a vario Max CN
analyser (Elementar Analysensysteme GmbH, Germany).
Statistics
The forest stands were organized into age classes: 0–1, 5–
10, 11–20, 21–30, 31–40, 41–50, 51–90 and 91–130 yr
(n= 5), and variations in production of extramatrical
hyphae in mesh bags, soil fungal biomass and nutrient avail-
abilities between age classes were analysed with one-way
ANOVA and Fishers LSD to separate the means. For each
stand, Shannon’s diversity index was calculated for EM
communities in the ingrowth bags as H¢=)Rp
i
log
e
p
i
,
where p
i
is the proportion of EM amplicons accounted for
by cluster i. Relationships between EM diversity and stand
age as well as C : N ratio and stand age were tested for sig-
nificance by linear regression. The relationship between
stand age and over-all distribution of EM amplicon between
clusters was tested for statistical significance by canonical
correspondence analysis (CCA), with stand age as a contin-
uous explaining variable. The analysis was repeated with
clusters merged within fungal genera. Relationships
between stand age and the relative abundance of amplicons
of individual EM clusters and genera were tested by
Spearman’s rank correlation. The CCA was conducted
using canoco (Microcomputer Power, Ithaca, NY, USA),
and all other statistical analyses were performed using the
software statistica (StatSoft Inc., Tulsa, OK, USA). A
species accumulation curve with stands as replicate samples
was produced, using the software estimates (Colwell, RK,
University of Connecticut, Storrs, CT, USA), and the
asymptote was estimated by the Chao2 method.
Results
Most of the mesh bags contained visible fungal mycelium,
except those collected from stands aged 0–1 yr (Fig. 1a).
The values of free and total ergosterol in the mesh bags were
similar in all age classes except for 30–40 yr, where the total
ergosterol was higher than the free ergosterol (Fig. 1b). The
age of the stand had a strong influence on hyphal growth in
the mesh bags, showing a peak in young stands before the
first thinning phase, when the trees were < 30 yr old
(ANOVAs for free and total ergosterol, and for EM
mycelial volume: P< 0.001, Fig. 1)
Useful PCR products were obtained from 28 of the 40
forest stands, with most of the unsuccessful samples
originating from recent clearcuts or very old stands and con-
taining little mycelium. We only obtained PCR products
from a single clearcut stand (0–1 yr) and from two old-
growth stands (90–130 yr). The numbers of replicates from
the other age classes are reported in Fig. 2. Sequencing
yielded a little over 18 000 ITS sequences; the number of
sequences per sample ranged from 300 to 1100. These val-
ues do not reflect the full potential of the 454 method, as a
great deal of the information consisted of nonITS
sequences, mainly primer dimers. The 18 000 sequences
were arranged in 248 clusters containing 99% of the total
number of sequences. Each of the remaining 184 sequences
occurred only once in the data set and were therefore dis-
missed as unreliable.
In most samples, EM fungi dominated the communities.
Averaged across all samples, 71% of the 18 000 amplicons
could be attributed to known EM taxa. By contrast, EM
fungi accounted for only 53 (21%) of the 248 clusters,
0
10
20
30
40
50
60
Volume of mycelium (µl g–1)
Age of forest (yr)
a
c
bc
ab
a
ab a
a
0
0.05
0.1
0.15
0.2
0.25
0.3
0–1 5–10 10–20 20–30 30–40 40–50 50–90 90–130
0–1 5–10 10–20 20–30 30–40 40–50 50–90 90–130
Ergosterol (µg g–1 sand)
Age of forest (yr)
bcd
x
a
yz
d
z
cd
z
bc
z
b
y
bcd yz
b
y
(a)
(b)
Fig. 1 Amount of ectomycorrhizal (EM) mycelium in sand from
mesh bags incubated in forests of different ages. (a) Visual estimates
of EM volume collected from mesh bags. (b) Free ergosterol (closed
bars) and total ergosterol (open bars). Error bars represent SE.
Different letters represents statistically significant differences
between treatments, n= 5 for each age group, (abc were used to
distinguish volume of mycelium in (a), as well as free ergosterol in
(b); xyz were used to distinguish total ergosterol in (b)).
New
Phytologist Research 1127
The Authors (2010)
Journal compilation New Phytologist Trust (2010)
New Phytologist (2010) 187: 1124–1134
www.newphytologist.com

indicating a long tail of rare nonmycorrhizal fungi. Four
samples from stands 0, 6, 110 and 124 yr of age deviated in
that the proportions of amplicons from known EM fungi
were low (0–30%). These samples also had a low content of
ergosterol. As the youngest and oldest age classes were repre-
sented by only one and two successfully amplifying samples,
respectively, and the proportion of EM amplicons in these
samples was low, these samples were excluded from further
analyses. After exclusion of these samples, the average pro-
portion of amplicons from EM taxa was 78% (median
86%). Among the amplicons from EM taxa, members of
the family Atheliaceae dominated. The most common
cluster was attributed to Tylospora fibrillosa, which was the
most common species in all age classes between 10 yr and
90 yr, and almost completely dominated among the EM
amplicons in 10- to 30-yr-old stands (Fig. 2). Amplicons
attributed T. asterophora, an as yet unidentified atheloid
species and various Piloderma species were also common. In
the CCA, stand age explained 7.1% of the eigenvalue of the
data, and the relationship was marginally insignificant
(P= 0.063) according to a Monte Carlo test with 1000
permutations. When clusters were merged within fungal
genera, 10.3% of the inertia was explained by stand age and
the relationship was significant (P= 0.028). The amplicon
abundances of the genera Xerocomus (P= 0.0006) and
Russula (P= 0.008), as well as the species Tylopilus felleus
(P= 0.015) and Byssocorticium pulchrum (P= 0.046), were
significantly and positively correlated with stand age.
Among the nonEM or nonassigned fungi, Sordariomycetes
and Leotiomycetes predominated, accounting for on average
8% and 6%, respectively, of the amplicons.
Both the number of EM clusters (P= 0.01) and
Shannon’s index based on EM taxa (P= 0.002) increased
significantly with stand age (Fig. 3). The species
0
20
40
60
80
100%
0–1 5–10 10–20 20–30 30–40 40–50 50–90 90–130
Age of forests (yr)
Amanita muscaria
UnID Atheliales
Russula emetica/betularum
Amanita fulva
Cortinarius sp.
UnID Atheliales
Russula ochroleuca
Tomentella terrestris
Lactarius necator
Piloderma sp.3
Xerocomus pruinatus
Amphinema byssoides
Russula paludosa
Piloderma fallax
Russula densifolia
Piloderma sp.2
Tomentellopsis sp.
UnID Atheliales
Pseudotomentella sp.3
Hygrophorus olivaceoalbus
Russula aquosa
Elaphomyces muricatus
Cortinarius biformis (b)
Pseudotomentella tristis
Cortinarius biformis (a)
Cortinarius semisanguineus
Amanita porphyria
Paxillus involutus
Tomentellopsis submollis
Inocybe napipes
Pseudotomentella mucidula
Tylopilus felleus
Cenococcum geophilum (b)
Inocybe stellatospora/lanuginos
a
Pseudotomentella sp.2
Cantharellus tubaeformis
Russula nigricans
Pseudotomentella sp.1
Piloderma sp.1
Cenococcum geophilum (a)
Wilcoxina sp.2
Wilcoxina sp.1
Tomentellopsis sp.1
Cortinarius rubellus
Byssocorticium pulchrum
Tomentella radiosa/sublilacina
Piloderma byssinum
Piloderma sphaerosporum
Xerocomus badius
Piloderma olivaceum
UnID Atheliales
Tylospora asterophora
Tylospora fibrillosa
ND ND
n = 3 n = 5 n = 4 n = 5 n = 3 n = 5
Proportion of EM amplicons
Fig. 2 Species distribution of internal transcribed spacer (ITS) amplicons from ectomycorrhizal (EM) fungi colonizing ingrowth mesh bags. The
mesh bags were incubated in the humus layer of Norway spruce (Picea abies) stands of different ages.
1128 Research
New
Phytologist
The Authors (2010)
Journal compilation New Phytologist Trust (2010)
New Phytologist (2010) 187: 1124–1134
www.newphytologist.com

accumulation curve, based on the stands as sampling units,
levelled off with increasing number of stands sampled, indi-
cating that the number of samples collected was sufficient to
describe most of the EM species colonizing the ingrowth
bags in the area (Fig. 4). The Chao2 estimate of the total
EM species richness was 65 (57–89 at the 95% confidence
interval).
When the composite soil samples were analysed, neither
the total ergosterol nor the free ergosterol concentration
varied significantly with tree age, in the humus layer or
the mineral soil. The concentration of free ergosterol was
significantly lower (P< 0.001) than that of total ergosterol
in both the humus (41% ± 2) and the upper mineral soil
(20 ± 2%, Fig. 5).
0306090
0
3
6
9
12
15
0306090
0
0.5
1.0
1.5
2.0
2.5
Age of forest (yr)
Age of forest (yr)
Number of EM clusters
Shannon index
(a)
(b)
Fig. 3 Diversity of ectomycorrhizal (EM) fungi in relation to age of
Norway spruce (Picea abies) stands: (a) number of EM clusters; (b)
Shannon diversity index.
0 5 10 15 20 25
0
10
20
30
40
50
60
70
Number of EM clusters
Number of stands
Fig. 4 Species accumulation curve (solid line) with 95%
confidence interval (fine broken lines). The uppermost broken
line indicates Chao2 estimated total species richness of
ectomycorrhizal (EM) fungi in Norway spruce (Picea abies)
stands.
0
20
40
60
80
100
120
0–1 5–10 10–20 20–30 30–40 40–50 50–90 90–130
0–1 5–10 10–20 20–30 30–40 40–50 50–90 90–130
Ergosterol in humus (µg g l–1)
Age of forest (yr)
0
0.5
1
1.5
2
2.5
3
3.5
4
Ergosterol in mineral soil (µg g–1)
A
g
e of forest (yr)
(a)
(b)
Fig. 5 Free (closed) and total (open) ergosterol
concentrations in soil samples collected from Norway spruce
(Picea abies) stands of different ages: (a) humus layer, (b) mineral
soil.
New
Phytologist Research 1129
The Authors (2010)
Journal compilation New Phytologist Trust (2010)
New Phytologist (2010) 187: 1124–1134
www.newphytologist.com

Nutrient availability in the soil varied to some extent with
stand age. Ammonium levels were significantly higher in
clearcuts compared with standing forests (Table 1). A
negative relationship was found between EM mycelial pro-
duction and NH
4
availability in young forests (0–36 yr,
Fig. 6) but not in older forests. The available PO
4
peaked in
clearcuts and both PO
4
and K peaked in the 30–40 yr old
forests. The availability of Ca did not vary significantly with
stand age (Table 1). The average C : N ratio in the humus
was 26.9 ± 0.4, and was positively related to stand age
(P= 0.025, Fig. 7). pH values showed maxima in clearcuts
(Table 1).
Discussion
The majority of the amplicons from fungi colonizing the
mesh bags were of EM origin. Although many saprotrophic
species were detected, they constituted a minor portion of
the amplicons.
In accordance with our expectations, the results indicated
a maximal EM mycelial production in young forests (10–
0
0.2
0.4
0.6
0.8
1
00.05 0.1 0.15 0.2 0.25 0.3 0.35
NH4 concentration (mg l–1)
Ergosterol in mesh bags (µg g–1 sand)
Fig. 6 Relationship between ectomycorrhizal (EM) growth and
NH
4
concentration in young Norway spruce (Picea abies) forests
(0–36 yr), y=)0.026–0.07log(x), r
2
= 0.43.
20
22
24
26
28
30
32
0 20 40 60 80 100 120 140
C:N ratio of humus
Age of forest (yr)
Fig. 7 Carbon (C) : nitrogen (N) ratio in the humus layer in relation
to Norway spruce (Picea abies) stand age. y= 25.8 + 0.25x,
r
2
= 0.12, P= 0.025.
Table 1 Effects of stand age on extractable nutrients and pH in the soil
Parameter 0–1 yr 5–10 yr 10–20 yr 20–30 yr 30–40 yr 40–50 yr 50–90 yr 90–130 yr
NH
4
0.53 ± 0.09a 0.24 ± 0.07b 0.17 ± 0.02b 0.20 ± 0.04b 0.27 ± 0.07b 0.21 ± 0.06b 0.21 ± 0.06b 0.13 ± 0.02b
PO
4
0.085 ± 0.009ab 0.061 ± 0.006bc 0.047 ± 0.007c 0.053 ± 0.011c 0.095 ± 0.02a 0.047 ± 0.004c 0.042 ± 0.015c 0.031 ± 0.004c
K 0.27 ± 0.02b 0.22 ± 0.04b 0.29 ± 0.04b 0.31 ± 0.03b 0.42 ± 0.06a 0.27 ± 0.02b 0.27 ± 0.02b 0.25 ± 0.03b
Ca (ns) 1.3 ± 0.3 1.6 ± 0.2 1.1 ± 0.2 1.0 ± 0.2 1.6 ± 0.5 1.3 ± 0.06 0.7 ± 0.2 0.8 ± 0.1
pH 4.3 ± 0.1a 4.1 ± 01ab 3.8 ± 0.1c 3.7 ± 0.1c 4.0 ± 0.1bc 3.9 ± 0.2bc 3.7 ± 0.1c 3.7 ± 0.1c
Values are given as mean ± SE, all values are mg g
)1
DW, except pH. Different letters within each row indicate statistically different values (P< 0.05). ns, not significant [correction added on
21 June 2010, after first online publication: the table formatting has been changed to aid clarity].
1130 Research
New
Phytologist
The Authors (2010)
Journal compilation New Phytologist Trust (2010)
New Phytologist (2010) 187: 1124–1134
www.newphytologist.com

20 yr), coinciding with canopy closure when tree growth is
rapid and leaf area maximal (Simard et al., 2004). Our
observations support the results of Palfner et al. (2005) and
Twieg et al. (2007), who found that the ratio of active to
senescent mycorrhizal root tips was higher in young than in
old forests. Palfner et al. (2005) estimated that 4 ·10
5
EM
root tips were active per m
2
in a 12-yr-old Sitka spruce for-
est, and that this decreased to 1 ·10
5
m
)2
in a 40-yr-old
forest, although the total number of root tips was similar.
Our findings suggest that much less C is required to sup-
port EM hyphal growth in very young and very old Norway
spruce forests, than during the period before the first thin-
ning, when C requirements appear to peak. A better
description of this variation in C allocation to root growth
and growth of EM hyphae over the rotation period is neces-
sary in order to improve C allocation models of forest
ecosystems, which are usually not well developed in this
respect (Litton & Giardina, 2008).
The extent to which the peak in belowground C flux in
young forests is associated with a peak in C sequestration in
the soil is not known. It is difficult to quantify C sequestra-
tion over the rotation period because of the large pool of
soil C and the small changes that occur during a single for-
est generation. King et al. (2007) reasoned that the much
higher root to shoot C ratio found in young stands of Pinus
resinosa (0.80 in 8-yr-old and 0.29 in 55-yr-old stands)
should have considerable implications for belowground C
accumulation. However, Vesterdal et al. (1995) found no
increase in total C accumulation after afforestation of for-
mer arable land, although soil C was redistributed from the
mineral soil to the forest floor over a period of 29 yr of
Norway spruce growth. The contribution of EM fungi to
this process was not investigated. The growth of external
EM mycelia did not influence the amount of C remaining
in litter over a 2-yr period in a study by Langley et al.
(2006). However, EM roots were degraded more slowly
than nonmycorrhizal roots, suggesting that the mycorrhizal
status of roots can have a strong influence on soil C process-
ing rates (Langley & Hungate, 2003).
Although the total species richness may be high, EM
communities associated with root tips are usually domi-
nated by a few species (Dahlberg, 2001; Horton & Bruns,
2001). In the present study, the EM community of external
mycelia was dominated by fast-growing T. fibrillosa, which
constituted 80% of the EM amplicons in the mesh bags
collected from 10- to 30-yr-old stands, but declined to an
average of 43% in mesh bags collected from 30- to 90-yr-
old stands. As the dominance of T. fibrillosa decreased, the
diversity of EM fungi in the mesh bags increased (Figs 2,
3). The same pattern was observed by Palfner et al. (2005)
in a Sitka spruce forest in northern England, where
T. fibrillosa colonized over 90% of the root tips in young
forests (1–6 yr), but only 40% of the root tips in 40-yr-old
forest. In Canada, Twieg et al. (2007) found increasing
diversity of EM fungi on root tips in Douglas-fir but not in
paper birch forests. The EM diversity stabilized after
c. 65 yr in the Douglas fir forest.
In line with the theories of Grime (1979), T. fibrillosa
might be described as a C strategist, being adapted to high
population densities. Carbon strategists are characterized by
efficient conversion of resources to biomass, leading to rapid
growth and ecosystem dominance when resources are abun-
dant. This is in agreement with the observed increase in
dominance of Tylospora species in ingrowth bags in
response to elevated atmospheric CO
2
concentrations
(Parrent & Vilgalys, 2007), which presumably increases
belowground allocation of photosynthates. It has been
shown in microcosm experiments that species with ample
production of extraradical mycelium may exclude other EM
species from the root systems of the host plants (Wu et al.,
1999), decreasing diversity. The competitive advantage of
T. fibrillosa may have declined in the maturing forest,
leaving room for other species, such as X. badius and B.
pulchrum to enter the community. We also found increases
in russuloid species (e.g. Russula nigricans) with stand age,
which confirms the results of other studies (Visser, 1995;
Smith et al., 2002; Kranabetter et al., 2005; Twieg et al.,
2007).
The mesh bag method may underestimate EM produc-
tion (Hendricks et al., 2006), presumably as a result of
discrimination against EM species belonging to the contact
or short distance exploration types according to the classifi-
cation of Agerer (2001). Furthermore, it has been shown
that species belonging to the genus Cortinarius are under-
represented in ingrowth bags, while boletoid species can be
over-represented compared with the EM community on
root tips (Kjo
¨ller, 2006). It should, however, be noted that
we detected several species of Cortinarius in the mesh bags
in the present study, although always in low amounts. On
the other hand, the use of ingrowth mesh bags made it
possible to sample the active portion of the EM community
from 40 Norway spruce stands with a limited amount of
effort. Studies of EM communities on root tips may include
inactive EM fungi and such studies are particularly labori-
ous because of the large number of root tips present in the
soil. The spatial variation of EM fungi was accounted for by
placing several mesh bags in each stand and pooling the
samples before analysis. According to the species accumula-
tion curve, most of the variation in the community of EM
fungi that colonized the mesh bags was reflected in our
measurements (Fig. 4).
Ammonium availability in the soil was negatively related
to hyphal growth in mesh bags in young forests, probably as
a result of efficient nutrient uptake by EM mycelium during
the period of most active growth. If this nutrient-absorbing
capacity is impaired, the risk of nutrient losses from the
system will increase. Large amounts of nitrate were lost from
Douglas fir stands in France (Marques et al., 1997; Jussy
New
Phytologist Research 1131
The Authors (2010)
Journal compilation New Phytologist Trust (2010)
New Phytologist (2010) 187: 1124–1134
www.newphytologist.com

et al., 2000) and Sitka spruce forests in Wales (Harrison
et al., 1995), probably as a result of impaired growth of
extramatrical hyphae owing to elevated N input. Nilsson
et al. (2007) found a negative relationship between hyphal
production in mesh bags and nitrate loss in oak stands along
a N deposition gradient in southern Sweden.
The observed progressive increase in C : N ratio with
stand age is in agreement with results from an 8000 yr long
chronosequence (Wallander et al., 2009) as well as with the
observed increasing C : N ratios in humus layers of increas-
ing age and depth (Lindahl et al., 2007). The increase may
be explained by selective uptake of N by mycorrhizal fungi,
with subsequent translocation to host tissue.
The trend towards a reduction in the proportion of free
ergosterol in older soil organic matter (SOM) is interesting,
and may have consequences for the way in which the
accuracy of fungal biomass estimates in soil is evaluated. In
newly formed young mycelia almost all the ergosterol was
in free form. By contrast, in the oldest SOM from the
mineral horizon, only 20% of the ergosterol was in free
form. This indicates that a portion of ergosterol becomes
bound in older SOM, and that this portion is unlikely to be
associated with active fungi. Another interesting finding was
that the concentration of free ergosterol (but not bound
ergosterol) in soil samples tended to be lower in clearcuts
than in young plantations. This may be the result of degra-
dation of EM mycelia from the previous forest generation
after cutting the forest. de Ridder-Duine et al. (2006)
found that free ergosterol constituted a small portion of the
total ergosterol in a forest soil with a high SOM content,
while most ergosterol was in free form in agricultural soils
with a lower SOM content. Ergosterol from inactive fungi
may thus be conserved in soils rich in SOM. The amount
of fungal biomass produced in the mesh bags was much
smaller than the fungal biomass estimated in the soil,
which would suggest an unrealistically long turnover
time, as discussed in Wallander et al. (2004). This is proba-
bly a result of underestimation of the EM production in the
mesh bags, as suggested by Hendricks et al. (2006) but
is should be noted that the calculated turnover time will
be reduced by using the free ergosterol rather than total
ergosterol.
In conclusion, the hyphal growth in mesh bags incubated
in Norway spruce forests in southern Sweden shows a
distinctive peak in young spruce stands that has not entered
the first thinning phase (< 30 yr old). This coincides with
the period of most active growth of fine roots. A single
species, T. fibrillosa, accounted for 80% of the amplicons in
the mesh bags during this period. The growth of T.
fibrillosa declined after 25 yr when other species such as
Xerocomus spp., Russula spp. and B. pulchrum became more
active. This massive growth of EM fungi in young forests
suggests that this period may be important for soil C
sequestration, although more research is required to clarify
the residence time of EM-derived C sources in the soil.
Acknowledgements
Funding was provided by The Swedish Research Council
for Environment, Agricultural Sciences and Spatial
Planning (Formas), the Swedish Energy Agency and So
¨dra’s
Foundation for Research, Development and Education.
References
Acosta-Martinez V, Dowd S, Sun Y, Allen V. 2008. Tag-encoded
pyrosequencing analysis of bacterial diversity in a single soil type as
affected by management and land use. Soil Biology & Biochemistry 40:
2762–2770.
Agerer R. 2001. Exploration types of ectomycorrhizae. Mycorrhiza 11:
107–114.
Altschul SF, Madden TL, Scha
¨ffer AA, Zhang J, Zhang Z, Miller W,
Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation
of protein database search programs. Nucleic Acids Research 25: 3389–
3402.
Bonet JA, Fisher CR, Colinas C. 2004. The relationship between forest
age and aspect on the production of sporocarps of ectomycorrhizal fungi
in Pinus sylvestris forests of the central Pyrenees. Forest Ecology and
Management 203: 157–175.
Bue
´e M, Reich M, Murat C, Morin E, Nilsson RH, Uroz S, Martin F.
2009. 454 Pyrosequencing analyses of forest soils reveal an unexpectedly
high fungal diversity. New Phytologist 184: 449–456.
Chisilov G, Demidova N 1998. Non-wood forest products and their
research in Arkhangelsk, Russia. In: Lund G, Pajari B, Korhonen M,
eds. European Forestry Institute Proceedings 23: 147–153.
Dahlberg A. 2001. Community ecology of ectomycorrhizal fungi: an
advancing interdisciplinary field. New Phytologist 150: 555–562.
Deacon JW, Flemming LV. 1992. Specificity phenomena in mycorrhizal
symbiosis: community-ecological consequences and practical
implications. In: Allen MF, ed. Mycorrhizal functioning: an integrating
plant fungal process. New York, NY, USA: Chapman & Hall, 249–300.
Edgar RC. 2004. Muscle: multiple sequence alignment with high accuracy
and high 16 throughput. Nucleic Acids Research 32: 1792–1797.
Fransson PMA, Taylor AFS, Finlay RD. 2001. Elevated atmospheric CO
2
alters root symbiont community structure in forest trees. New Phytologist
152: 431–442.
Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for
basidiomycetes - application to the identification of mycorrhizae and
rusts. Molecular Ecology 2: 113–118.
Grime JP. 1979.Plant strategies and vegetation processes. New York, NY,
USA: John Wiley.
Harrison AF, Stevens PA, Dighton J, Quarmby C, Dickinson AL, Jones
HE, Howard DM. 1995. The critical load of nitrogen for Sitka spruce
forests on stagnopodsols in Wales; role of nutrient limitations. Forest
Ecology and Management 76: 139–148.
Hedh J, Wallander H, Erland S. 2008. Ectomycorrhizal mycelial
species composition in apatite amended and non-amended mesh
bags buried in a phosphorus-poor spruce forest. Mycological Research
112: 681–688.
Hendricks JJ, Mitchell RJ, Kuehn KA, Pecot SD, Sims SE. 2006.
Measuring external mycelia production of ectomycorrhizal fungi in the
field: the soil matrix matters. New Phytologist 171: 179–186.
Hintikka V 1988. On the macromycete flora on oligotrophic pine forest of
different ages in South Finland. Acta Botanica Fennica 136: 95–98.
1132 Research
New
Phytologist
The Authors (2010)
Journal compilation New Phytologist Trust (2010)
New Phytologist (2010) 187: 1124–1134
www.newphytologist.com

Ho
¨berg P, Nordgren A, Buchmann N, Taylor AFS, Ekblad A, Ho
¨gberg
MN, Nyberg G, Ottoson-Lovenius M, Read DJ. 2001. Large-scale
forest girdling shows that current photosynthesis drives soil respiration.
Nature 411: 789–792.
Horton TR, Bruns TD. 2001. The molecular revolution in
ectomycorrhizal ecology: peeking into the black box. Molecular Ecology
10: 1855–1871.
Jumpponen A, Trappe JM, Cazares E. 1999. Ectomycorrhizal fungi in
Lyman Lake Basin: a comparison between primary and secondary
successional sites. Mycologia 91: 575–582.
Jussy J-H, Colin-Belgrand M, Ranger J. 2000. Production and root
uptake of mineral nitrogen in a chronosequence of Douglas-fir
(Pseudotsuga menziesii) in the Beaujolais Mounts. Forest Ecology and
Management 128: 197–209.
King JS, Giardina CP, Pregitzer KS, Friend AL. 2007. Biomass
partitioning in red pine (Pinus resinosa) along a chronosequence in the
Upper Peninsula o Michigan. Canadian Journal of Forest Research 37:
93–102.
Kjo
¨ller R. 2006. Disproportionate abundance between ectomycorrhizal
root tips and their associated mycelia. FEMS Microbiology Ecology 58:
214–224.
Ko
¨ljalg U, Larsson KH, Abarenkov K, Nilsson RH, Alexander IJ,
Eberhardt U, Erland S, Hoiland K, Kjoller R, Larsson E et al. 2005.
UNITE: a database providing web-based methods for the molecular
identification of ectomycorrhizal fungi. New Phytologist 166: 1063–
1068.
Korkama T, Fritze H, Pakkanen A, Pennanen T. 2007. Interactions
between extraradical ectomycorrhizal mycelia, microbes associated with
the mycelia and growth rate of Norway spruce (Picea abies) clones. New
Phytologist 173: 798–807.
Kranabetter JM, Friesen J, Gamiet S, Kroeger P. 2005. Ectomycorrhizal
mushroom distribution by stand age in western hemlock-lodgepole pine
forest in northwestern British Colombia. Canadian Journal of Forest
Research 35: 1527–1539.
Langley JA, Chapman SK, Hungate BA. 2006. Ectomycorrhizal
colonization slows root decomposition: the post-mortem fungal legacy.
Ecology Letters 9: 955–959.
Langley JA, Hungate BA. 2003. Mycorrhizal controls on belowground
litter quality. Ecology 84: 2302–2312.
Last FT, Dighton J, Mason PA. 1987. Succession of sheathing
mycorrhizal fungi. Trends in Ecology and Evolution 2: 157–161.
Lindahl BD, Ihrmark K, Boberg J, Trumbore SE, Ho
¨gberg P, Stenlid J,
Finlay RD. 2007. Partial separation of litter decomposition and
mycorrhizal nitrogen uptake in a boreal forest. New Phytologist 173:
611–620.
Litton CM, Giardina CP. 2008. Below-ground carbon flux and
partitioning: global patterns and response to temperature. Functional
Ecology 22: 941–954.
Majdi H, Truus L, Johansson U, Nylund JE, Wallander H. 2008.
Effects of slash retention and wood ash addition on fine root
biomass and production and ectomycorrhizal mycelium in a Norway
spruce stand in SW Sweden. Forest Ecology and Management 255:
2109–2117.
Malmstro
¨m C. 1937.To
¨nnersjo
¨hedens fo
¨rso
¨kspark i Halland.
Meddelanden fra
˚n Statens Skogsfo
¨rso
¨ksanstalt 30–3: 323–528. (In
Swedish with German summary).
Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA,
Berka J, Braverman MS, Chen Y-J, Chen Z et al. 2005. Genome
sequencing in microfabricated high-density picolitre reactors. Nature
437: 376–380.
Marques R, Ranger J, Villette S, Granier A. 1997. Nutrient dynamics in a
chronosequence of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco)
stands on the Beaujolais Mounts (France). 2. Quantitative approach.
Forest Ecology and Management 92: 167–197.
Murphy J, Riley JP. 1962. A modified single solution method for the
determination of phosphate in natural waters. Analytica Chimica Acta
27: 31–36.
Nilsson LO, Falkengren-Grerup U, Ba
˚a
˚th E, Wallander H. 2007.N
deposition in oak forests reduces mycelial growth by ectomycorrhizal
fungi, while other soil microbes are more influenced by soil pH.
Oecologia 153: 375–384.
Nylund J-E, Wallander H. 1992. Ergosterol analysis as a means of
quantifying mycorrhizal biomass. In: Norris JR, Read DJ, Varma AK,
eds. Methods in microbiology vol. 24. London, UK: Academic Press,
77–88.
Palfner G, Casanova-Katny MA, Read DJ. 2005. The mycorrhizal
community in a forest chronosequence of Sitka spruce Picea sitchensis
(Bong.) Carr. in northern England. Mycorrhiza 15: 571–579.
Parrent JL, Vilgalys R. 2007. Biomass and compositional response of
ectomycorrhizal fungal hyphae to elevated CO
2
and nitrogen
fertilization. New Phytologist 176: 164–174.
de Ridder-Duine AS, Smant W, van der Wal A, van Veen JA, de Boer W.
2006. Evaluation of a simple, non-alkaline extraction protocol to
quantify soil ergosterol. Pedobiologia 50: 293–300.
Schmalholz M, Hylander K. 2009. Succession of bryophyte assemblages
following clear-cut logging in boreal spruce-dominated forests in south-
central Sweden – does retrogressive succession occur? Canadian Journal
of Forest Research 39: 1871–1880.
Simard SW, Jones MD, Durall DM. 2002. Carbon and nutrient fluxes
within and between mycorrhizal plants. In: van der Heijden MGA,
Sanders IR, eds. Mycorrhizal ecology. Berlin, Germany: Springer, 33–
74.
Simard SW, Sachs DL, Vyse A, Blevins LL. 2004. Paper birch competitive
effects vary with conifer tree species and stand age in interior British
Colombia forests, implications for reforestation policy and practice.
Forest Ecology and Management 198: 55–74.
Smith JA, Moo
¨ina R, Huso MMP, Luoma DL, McKay D, Castellano
MA, Lebel T, Valachovic Y. 2002. Species richness, abundance and
composition of hypogeous and epigeous ectomycorrhizal fungal
sporocarps in young, rotation-age and old-growth stands of Douglas-fir
(Pseudotsuga menziesii) in the Cascade Range of Oregon, USA.
Canadian Journal of Botany 80: 186–204.
Smith SE, Read DJ. 2008.Mycorrhizal symbiosis,3rd edn. London, UK:
Academic Press.
Twieg BD, Durall DM, Simard SW. 2007. Ectomycorrhizal fungal
succession in mixed temperate forests. New Phytologist 176: 437–447.
Vesterdal L, Dalsgaard M, Felby C, Raulund-Rasmussen K, Jo
¨rgensen
BB. 1995. Effects of thinning and soil properties on accumulation of
carbon, nitrogen and phosphorus in the forest floor of Norway spruce
stands. Forest Ecology and Management 77: 1–10.
Visser S. 1995. Ectomycorrhizal fungal succession in jack pine stands
following wildfire. New Phytologist 129: 389–401.
Wallander H, Go
¨ransson H, Rosengren U. 2004. Production, standing
biomass and natural abundance of
15
N and
13
C in ectomycorrhizal
mycelia collected at different soil depths in two forest types. Oecologia
139: 89–97.
Wallander H, Mo
¨rth C-M, Giesler R. 2009.
15
N enrichment in soil profiles
in relation to microbial community composition along a chronosequence
in the upheaval region of northern Sweden. Oecologia 169: 87–96.
Wallander H, Nilsson LO, Hagerberg D, Ba
˚a
˚th E. 2001. Estimation of
the biomass and seasonal growth of external mycelium of
ectomycorrhizal fungi in the field. New Phytologist 151: 753–760.
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis
MA, Gelfland DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to
methods and applications. San Diego, CA, USA: Academic Press, 315–322.
Wu B, Nara K, Hogetsu T. 1999. Competition between ectomycorrhizal
fungi colonizing Pinus densiflora.Mycorrhiza 9: 151–159.
New
Phytologist Research 1133
The Authors (2010)
Journal compilation New Phytologist Trust (2010)
New Phytologist (2010) 187: 1124–1134
www.newphytologist.com

Yuan J-P, Kuang H-C, Wang J-H, Liu X. 2008. Evaluation of ergosterol
and its esters in the pileus, gill, and stipe tissues of agaric fungi and their
relative changes in the comminuted fungal tissues. Applied Genetics and
Molecular Biotechnology 80: 459–465.
Supporting Information
Additional supporting information may be found in the
online version of this article.
Table S1 Descriptions of the sites.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting information
supplied by the authors. Any queries (other than missing
material) should be directed to the New Phytologist Central
Office.
1134 Research
New
Phytologist
The Authors (2010)
Journal compilation New Phytologist Trust (2010)
New Phytologist (2010) 187: 1124–1134
www.newphytologist.com